Biotechnology includes the delivery of genetic information to a cell to express an exogenous nucleotide sequence, to inhibit, eliminate, augment, or alter expression of an endogenous nucleotide sequence, or to express a specific physiological characteristic not naturally associated with the cell. Polynucleotides may be coded to express a whole or partial protein, or may be anti-sense, or non-viral DNA, or recombine with chromosomal DNA.
A basic challenge for biotechnology and thus its subpart, gene therapy, is to develop approaches for delivering genetic information to cells of a patient in a way that is efficient and safe. This problem of drug delivery, where the genetic material is a drug, is particularly challenging. If genetic material are appropriately delivered they can potentially enhance a patient's health and, in some instances, lead to a cure. Therefore, a primary focus of gene therapy is based on strategies for delivering genetic material in the form of nucleic acids. After delivery strategies are developed they may be sold commercially since they are then useful for developing drugs.
Delivery of a nucleic acid means to transfer a nucleic acid from a container outside a mammal to near or within the outer cell membrane of a cell in the mammal. The term transfection is used herein, in general, as a substitute for the term delivery, or, more specifically, the transfer of a nucleic acid from directly outside a cell membrane to within the cell membrane. The transferred (or transfected) nucleic acid may contain an expression cassette. If the nucleic acid is a primary RNA transcript that is processed into messenger RNA, a ribosome translates the messenger RNA to produce a protein within the cytoplasm. If the nucleic acid is a DNA, it enters the nucleus where it is transcribed into a messenger RNA that is transported into the cytoplasm where it is translated into a protein. Therefore if a nucleic acid expresses its cognate protein, then it must have entered a cell. A protein may subsequently be degraded into peptides, which may be presented to the immune system.
It was first observed that the in vivo injection of plasmid DNA into muscle enabled the expression of foreign genes in the muscle (Wolff 1990). Since that report, several other studies have reported the ability for foreign gene expression following the direct injection of DNA into the parenchyma of other tissues. Naked DNA was expressed following its injection into cardiac muscle (Acsadi 1991).
The muscular dystrophies (MD) are a heterogeneous group of mostly inherited disorders characterized by progressive muscle wasting and weakness which eventually leads to death. In most in not all forms of MD, the disease is associated with either a non-functioning or malfunctioning protein due to the presence of a mutant or deleted gene (Hartigan-O'Connor 2000). Because of the nature of these diseases, few traditional treatments are available. However, because the genes and protein products that are responsible for most of the dystrophies have been identified, delivery of corrective genes offers a promising treatment.
Several attributes of striated muscle cells make genetic repair feasible. First, myofibers have a long life span, facilitating long term persistence of delivered genes. Second, for DMD and MCDM, it has been shown that gene replacement in striated muscle alone can alleviate the major features of the disease (Cox 1993, Kuang 1998). Third, dystrophin positive fibers may possess a survival advantage over dystrophin negative fibers, suggesting that only a portion of the fibers need to receive the correcting polynucleotide (Morgan 1993). Finally, only 20% of the normal level of dystrophin is required to be asymptomatic. Thus, low level dystrophin expression in a majority of muscle fibers may be sufficient for elimination of symptoms (Phelps 1995).